CN104357340A - Method for producing L-malic acid by virtue of biotransformation of fumaric acid - Google Patents
Method for producing L-malic acid by virtue of biotransformation of fumaric acid Download PDFInfo
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- CN104357340A CN104357340A CN201410648894.5A CN201410648894A CN104357340A CN 104357340 A CN104357340 A CN 104357340A CN 201410648894 A CN201410648894 A CN 201410648894A CN 104357340 A CN104357340 A CN 104357340A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
Abstract
The invention relates to a method for producing L-malic acid by virtue of biotransformation of fumaric acid. The method comprises the following steps of carrying out fermentative cultivation on autonomously selected clavispora lusitaniae CICC 33054 and centrifuging to obtain yeast cells; adding the yeast cells to a sodium fumarate liquid and carrying out biotransformation, replacing by virtue of calcium fumarate, precipitating, filtering, carrying out acidolysis by sulfuric acid, filtering, concentrating, crystallizing and drying to obtain the L-malic acid finished product. The clavispora lusitaniae CICC 33054 is easily cultured and high in transformation efficiency, when the clavispora lusitaniae CICC 33054 is used for the production of L-malic acid, the production efficiency can be increased and the production cost is reduced.
Description
Technical field
The present invention relates to a kind of method utilizing fumaric acid to produce L MALIC ACID, especially relate to the method that L MALIC ACID produced by Clavispora lusitaniae yeast CICC 33054 bio-transformation fumaric acid.
Background technology
L MALIC ACID has another name called hydroxy succinic acid or hydroxy-butanedioic acid, and molecular formula is C
4h
6o
5, molecular weight is 134.09.At normal temperatures, L MALIC ACID is white crystals or crystalline powder, odorless, has frank tart flavour, proportion 1.601, fusing point 98 ~ 99 DEG C.At 20 DEG C, the L MALIC ACID aqueous solution of 34%, without opticity, can occur left-handed after thin up, when its concentration is more than 34%, occurs dextrorotation again.
Owing to containing-OH base and-COOH base in L MALIC ACID molecule, therefore large to the solubleness of polarity solvent, chemical property is comparatively active.It is very easily dissolved in ethanol at normal temperatures, and in water, its solubleness increases with the rising of water temperature, in strongly-acid.It can also react with alcohols, the vitriol oil, phenol, hydrogen peroxide, trichoro-aldehyde and plumbic acetate etc., and energy and 2-Naphthol, a phenol and 2,7-naphthalenediol effects, make solution be distinct colors.
L MALIC ACID is the important organic acid produced in organism metabolic process.In microorganism, its generation is relevant with many important pathways metabolisms, and coming across in tricarboxylic acid cycle (TCA) and branch road glyoxylate cycle thereof, is also CO
2the product of fixation reaction.
In decades, people are studied the pathways metabolism of L MALIC ACID in microorganism, especially to Split-gill (
schizphylhls commne) research of pathways metabolism is more deep.The investigator being engaged in this respect work at present mostly thinks CO
2fixation reaction is the significant process that L MALIC ACID generates and accumulates.
L MALIC ACID solubleness in water is large, tart flavour is soft, local flavor is unique, quality is stable, and its purposes is quite extensive:
1. in foodstuffs industry, L MALIC ACID is a kind of security foodstuff additive of generally acknowledging in the world, is also a kind of excellent acidity regulator and preservation agent.Compared with citric acid, the stimulation of L MALIC ACID is comparatively slow, and tart flavour can retain the long period, with citric acid with the use of, the tart flavour feature of natural fruits can be simulated, sweet sour mouthfeel is seemed nature, plentiful, tuning.In the foodstuff production of present America and Europe and Japanese various countries, it has become one of indispensable basic raw material.In addition, pH value can be made to be adjusted owing to adding L MALIC ACID in food, add the anti-microbial effect that itself is intrinsic, L MALIC ACID is also widely used in other foodstuffs industry, as made the preservation agent etc. of fishery products.
2., pharmaceutically, L MALIC ACID has important physiological function.L MALIC ACID participates in body metabolism directly, helpful to the health of human body.Can be used for treating anaemia, hepatic insufficiency, liver failure, and the component of mixed amino acid injection and the tranquilizer of calcium lactate injection liquid can be made.L MALIC ACID add also can promote medicine in medicine stability, improve the receptivity of human body to medicine.L MALIC ACID potassium can be used as the important sources of potassium, and also tool prevents effect of oedema, hyperpiesia and fat generation.L MALIC ACID and its esters can also make the growth stimulant of animal.
3. other industrial purposes.Due to L MALIC ACID tool antioxygenation and stronger huge legendary turtle cooperation use, be therefore widely used in printing and dyeing industry as color preserving agent and synergistic agent.In chemical industry, due to the adjustable pH value of L MALIC ACID, therefore can do the seasonings of toothpaste and tobacco, skin cleaner, scolding tin soldering flux, air freshener, detergent builder and waste-gas desulfurization agent, citric acid also can be replaced as the rust remover on various metal vessel and surface.On building materials, add appropriate L MALIC ACID in cement, can setting time be shortened, prevent the generation of alkaline aggregation, improve concrete intensity.L MALIC ACID also can replace oxalic acid as the surface cleaning agent of various stone, makes its surface become smooth, smooth and attractive in appearance.
Along with deepening continuously of L MALIC ACID applied research, its market demand increases year by year.Through continuous effort, the production method of L MALIC ACID, from direct extraction method single in early days, develops into the multiple production method of today.According to the difference of raw materials for production, the production of L MALIC ACID can be divided into 5 kinds:
1. direct extraction method: milk of lime is directly added in the fruit juice and vegetables juice that people is rich in L MALIC ACID, the L MALIC ACID calcium deposit vitriolization of formation, and then reclaim free L MALIC ACID.
2. Split Method: under certain condition, alditol, β-Nei vinegar, cyclopentadiene and butene dioic acid etc. all can synthesize DL-oxysuccinic acid.The DL-oxysuccinic acid of chemosynthesis is split and just can obtain L MALIC ACID.
3. utilize microbial transformation fumaric acid to produce L MALIC ACID.L MALIC ACID is produced with the microbial transformation fumaric acid that can produce fumarase.Up to the present, screen the bacterial classification with higher fumarase activity have short lactobacillus (
lactobacillus brevis), Brevibacterium ammoniagenes (
brevibacterium ammoniagenes), brevibacterium flavum (
brevibacterium flavum), Paracoccus denitrificans (
paracoccus denitrificans), secondary entero-bacte (
porocolobactoriuo sp.), Candida lipolytica (
candida lipolytica) and Aspergillus wentti (
aspergillus wentii) etc.The method that biotransformation method adopts has immobilized enzyme or immobilized cell and free cell method.The enzyme of immobilized enzyme or immobilized cell reaction and cell can reuse, but immobilization complex operation, and equipment requirements is high.Free cell method technique simple to operate is easily controlled.
4. utilize fermentable saccharine material to produce L MALIC ACID.Adopt the production ways of this method to have two: Article 1 produces L MALIC ACID with single culture one-step fermentation, bacterial classification used have flavus (
aspergillus flavus), Aspergillus parasiticus (
aspergillus parasiticus), aspergillus oryzae (
aspergillus oryzae) and Aureobasidium pullulans (
aureobasidium pullulans) etc.; Article 2 be miscegenation ferment two steps produce L MALIC ACIDs, first adopt rhizopus arrhizus (
rhizopus arrizus) or zhizopchin (
rhizopus chinensis) saccharine material is changed into fumaric acid, then utilize Pichia membranaefaciens (
pichia membranaefaciens), proteus vulgaris (
proteus vulgaris) and paecilomyces varioti (
paecilomyces variotii) in one, fumaric acid is changed into L MALIC ACID.Because fermentation period is longer, acid production rate is relatively low, by product is more, therefore up to this point also there is not yet the report being applied to large-scale commercial production.
5. utilize the non-saccharine material of fermentable to produce L MALIC ACID.Current people successfully adopt Boulogne candiyeast (
candida brumptii) fermentation N PARAFFIN & HEAVY NORMAL PARAFFIN, paecilomyces varioti fermenting alcohol, glycerine, acetic acid and propionic acid etc., Alcaligenes (
alcaligenessp. T501) ferment toxilic acid, and its transformation efficiency reaches 80%, 48% and 98.4% respectively.But the test of these non-saccharine material fermentative production L MALIC ACIDs is still in laboratory stage at present, not yet throw people's industrial production.
Summary of the invention
Technical problem solved by the invention is for providing a kind of with the microbial strains Clavispora lusitaniae yeast CICC 33054(Clavispora lusitaniae CICC 33054 producing fumarase)) bio-transformation fumaric acid production L MALIC ACID.
The Clavispora lusitaniae yeast CICC 33054 of the autonomous seed selection of the present invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.9848.
The 26S rDNA sequence of Clavispora lusitaniae yeast CICC 33054 is shown in sequence table.
Clavispora lusitaniae yeast CICC 33054 fumarase vigor of the present invention is high, cultivates enzyme activity by fermentation and can reach 2000U/g wet cell.Produce L MALIC ACID for bio-transformation fumaric acid, transformation efficiency is high, is convenient to subsequent extracted, can enhance productivity, and reduces production cost.
The present invention further provides a kind of method that L MALIC ACID produced by microbial transformation fumaric acid.By the centrifugal acquisition yeast cell of Clavispora lusitaniae yeast CICC 33054 fermentation culture secondary fermentation liquid, add sodium fumarate solution, by the fumarase in somatic cells, optics specificity synthesis L MALIC ACID, then L MALIC ACID calcium deposit is washed to obtain through Fumaric acid, calcium salt cementation, filtration, again through sulphuric acid soln acidolysis, filter and wash to obtain L MALIC ACID solution, through concentrated, crystallization, dry L MALIC ACID finished product.
Concrete steps are:
(1) Clavispora lusitaniae yeast CICC 33054 slant strains inoculation seed culture medium fermentation culture 18 ~ 24h, obtains inoculation liquid;
(2) inoculation liquid access fermention medium, fermentation 20 ~ 30h obtains fermented liquid, fermented liquid centrifugal acquisition Clavispora lusitaniae yeast CICC 33054 cell;
(3) Clavispora lusitaniae yeast CICC 33054 cell adds sodium fumarate solution, and bio-transformation generates oxysuccinic acid, through precipitation, filtrations, acidolysis, filtration, concentrated, crystallization, dry must finished product L MALIC ACID sour.
The inclined-plane seed culture of Clavispora lusitaniae yeast CICC 33054 can adopt this area common technology means.Slant medium can adopt test tube or the preparation of eggplant bottle.Shake flask fermentation can be adopted under normal circumstances to cultivate and to obtain appropriate seed culture fluid, if need large scale culturing, fermentor tank enlarged culturing can be carried out again, the seed culture fluid that acquisition amount is large.In industrial production, Clavispora lusitaniae yeast CICC 33054 cell fermentation liquid carries out fermentation culture by fermentor tank.
Some typical substratum are composed as follows:
Slant medium (g/L): Fructus Hordei Germinatus leaching powder 80 ~ 150.0, agar 15.0, pH value 6.0 ~ 7.0.
Seed culture medium (g/L): glucose 30.0 ~ 70.0, yeast powder 2.0 ~ 8.0, ammonium sulfate 3.0 ~ 8.0, potassium primary phosphate 1.0 ~ 5.0, magnesium sulfate heptahydrate 0.1 ~ 0.6, regulates pH6.0 ~ 7.0.
Fermention medium (g/L): glucose 50.0 ~ 120.0, yeast powder 2.0 ~ 8.0, ammonium sulfate 3.0 ~ 8.0, potassium primary phosphate 1.0 ~ 5.0, magnesium sulfate heptahydrate 0.1 ~ 0.6, adjust ph 6.0 ~ 7.0.
Microorganism Deposit Information
Microorganism classification is named: Clavispora lusitaniae yeast (
clavispora lusitaniae)
Deposit number: CGMCC No.9848
Preservation date: on October 27th, 2014;
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center;
Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101.
embodiment
For further illustrating the present invention, illustrate with the following Examples.
Embodiment 1
Microbial strains is identified
1, extracting genome DNA
(1) by transfering loop picking one ring slant strains, be suspended in 1ml 0.85% NaCl solution, the centrifugal 2min of 10000r/min, abandons supernatant;
(2) precipitation is resuspended in 550ul 1 × TE, boiling water bath 10min;
(3) 3ul Proteinase K (20ug/ml) is added, 37 DEG C of incubation 30min;
(4) 30ul 10% SDS is added, 37 DEG C of incubation 30min;
(5) add 100ul 5M NaCl solution fully to mix, then add 80ul CTAB/NaCl and mix, 65 DEG C of water-bath 10min;
(6) add equal-volume chloroform/primary isoamyl alcohol (24:1), shake mixing gently, the centrifugal 8min of 10000r/min;
(7) get supernatant liquor, add equal-volume phenol/chloroform/primary isoamyl alcohol (25:24:1), concussion shakes up gently; The centrifugal 8min of 13000r/min;
(8) repeating step 6;
(9) get supernatant, add 0.6-0.8 times of volume isopropanol, after mixing, room temperature leaves standstill 30min gently;
(10) the centrifugal 8min of 10000r/min, abandons supernatant, adds 500ul 70% ethanol, puts upside down the desalinization of soil by flooding or leaching for several times, abandons supernatant; Repeat the desalinization of soil by flooding or leaching twice;
(11) centrifuge tube is inverted, dry to DNA precipitation; Precipitation is dissolved in 50-100ul dd H
2o ,-20 DEG C save backup.
2,26S rDNA increases
PCR reaction system 50ul:DNA masterplate 0.5ul(is about 10ng), 10 × Taq archaeal dna polymerase Buffer 5ul, 10pmol/ul dNTP 1ul, the forward and reverse primer of 10pmol/ul each 1ul(forward primer NL1 5 '-GCA TAT CAA TAA GCG GAG GAA AAG-3 ' and reverse primer NL4 5 '-GGT CCG TGT TTC AAG ACG G-3 '), 2.5u/ul Taq archaeal dna polymerase 0.5ul, adds ddH
2o to 50ul.Pcr amplification reaction program: 94 DEG C of 5min; 94 DEG C of 30s; 50 DEG C of 30s; 72 DEG C of 1min 30s; 35 cycles; 72 DEG C of 10min; 4 DEG C of preservations.Pcr amplification product 1% agarose gel electrophoresis detects.PCR primer ABI3700 gene sequencer checks order.
3, sequence alignment qualification
Microbial strains 26S rDNA sequence is in ncbi database comparison and type strain
clavispora lusitaniaecBS 4413
t(AY190538),
clavispora lusitaniaecBS 6936
t(U44817), similarity is the highest, is respectively 100.0%, 93.4%.Identification of strains called after Clavispora lusitaniae yeast CICC 33054(Clavispora lusitaniae CICC 33054).
Embodiment 2
Slant medium (g/L): Fructus Hordei Germinatus leaching powder 90.0, agar 15.0, pH value 6.0.
Seed culture medium (g/L): glucose 40.0, yeast powder 3.0, ammonium sulfate 4.0, potassium primary phosphate 2.0, magnesium sulfate heptahydrate 0.2, regulates pH6.0.
Fermention medium (g/L): glucose 70.0, yeast powder 6.0, ammonium sulfate 6.0, potassium primary phosphate 2.0, magnesium sulfate heptahydrate 0.2, regulates pH6.0.
Getting Clavispora lusitaniae yeast CICC 33054 slant strains one ring is inoculated in the triangular flask that 30mL seed culture medium is housed, shaking table 200rpm, cultivates 18h for 29 DEG C and obtains inoculation liquid.Get 3mL inoculation liquid, be inoculated in the triangular flask that 300mL fermention medium is housed, 200rpm cultivates 28h, obtain Clavispora lusitaniae yeast CICC 33054 fermented liquid, fermented liquid 5000rpm is centrifugal, and 10min obtains yeast cell, add the fumaric acid solution (sodium hydroxide adjust ph is 6.0) of the 139g/L of 300mL, then add 0.2mL toluene, 30 DEG C of bio-transformation 31h generate L MALIC ACID.Add Fumaric acid, calcium salt displacement, filter to obtain L MALIC ACID calcium deposit, then after sulfuric acid solution, filter to obtain L MALIC ACID solution, 80 DEG C concentrate, and obtain L MALIC ACID finished product after crystallisation by cooling, drying, purity reaches 99.8%.Concentrated mother liquor can be merged in next batch L MALIC ACID solution.
Case study on implementation 3
Slant medium (g/L): Fructus Hordei Germinatus leaching powder 150.0, agar 15.0, pH value 7.0.
Seed culture medium (g/L): glucose 70.0, yeast powder 8.0, ammonium sulfate 8.0, potassium primary phosphate 5.0, magnesium sulfate heptahydrate 0.6, adjust ph 7.0.
Fermention medium (g/L): glucose 120.0, yeast powder 15.0, ammonium sulfate 12.0, potassium primary phosphate 5.0, magnesium sulfate heptahydrate 0.6, adjust ph is 7.0.
Get Clavispora lusitaniae yeast CICC 33054 slant strains one ring to be inoculated into and to be equipped with in the 1L triangular flask of 300mL seed culture medium, shaking table 200rpm, cultivate 24h for 33 DEG C and obtain inoculation liquid, merge to obtain fermented liquid 600mL.Get 600mL inoculation liquid, be inoculated into the 50L fermentor tank that 30L fermention medium is housed, 33 DEG C, air flow 1v/v.min, 500rpm cultivate 24h, obtain Clavispora lusitaniae yeast CICC 33054 fermented liquid, fermented liquid 5000rpm is centrifugal, and 10min obtains yeast cell, add the fumaric acid solution (sodium hydroxide adjust ph is 6.0) of the 139g/L of 60L, then add 300ml toluene, 40 DEG C of bio-transformation 28h generate L MALIC ACID.Add Fumaric acid, calcium salt displacement, filter to obtain L MALIC ACID calcium deposit, then after sulfuric acid solution, filter to obtain L MALIC ACID solution, 80 DEG C concentrate, and obtain L MALIC ACID finished product after crystallisation by cooling drying, purity reaches 99.8%.Concentrated mother liquor can be merged in next batch L MALIC ACID solution.Adding Fumaric acid, calcium salt displacement filters the filtered liquid after L MALIC ACID calcium deposit can be used further to transform and produces L MALIC ACID.
Case study on implementation 4
Slant medium (g/L): Fructus Hordei Germinatus leaching powder 150.0, agar 15.0, pH value 7.0.
Seed culture medium (g/L): glucose 70.0, yeast powder 8.0, ammonium sulfate 8.0, potassium primary phosphate 5.0, magnesium sulfate heptahydrate 0.6, adjust ph 7.0.
Fermention medium (g/L): glucose 120.0, yeast powder 15.0, ammonium sulfate 12.0, potassium primary phosphate 5.0, magnesium sulfate heptahydrate 0.6, adjust ph is 7.0.
Get Clavispora lusitaniae yeast CICC 33054 slant strains one ring to be inoculated into and to be equipped with in the 1L triangular flask of 300mL seed culture medium, shaking table 200rpm, cultivate 24h for 33 DEG C and obtain inoculation liquid, merge to obtain fermented liquid 600mL.Get 600mL inoculation liquid, be inoculated into the 50L fermentor tank that 30L seed culture medium is housed, 33 DEG C, air flow 1v/v.min, 500rpm cultivate 20h, obtain Clavispora lusitaniae yeast CICC 33054 secondary seed solution.Get the access of 300L secondary seed solution and 15m is housed
320 m of fermention medium
3fermentor tank, 33 DEG C, air flow 0.5v/v.min, 200rpm cultivate 20h.The centrifugal 10min of fermented liquid obtains yeast cell, adds 30 m
3the fumaric acid solution (sodium hydroxide adjust ph is 6.0) of 139g/L, then add 400L toluene, 40 DEG C of bio-transformation 30h generate L MALIC ACIDs.Add Fumaric acid, calcium salt displacement, filter to obtain L MALIC ACID calcium deposit, then after sulfuric acid solution, filter to obtain L MALIC ACID solution, 80 DEG C concentrate, and obtain L MALIC ACID finished product after crystallisation by cooling drying, purity reaches 99.8%.Concentrated mother liquor can be merged in next batch L MALIC ACID solution.Adding Fumaric acid, calcium salt displacement filters the filtered liquid after L MALIC ACID calcium deposit can be used further to transform and produces L MALIC ACID.
Above-described case study on implementation is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under the prerequisite not departing from design spirit of the present invention; the various distortion that the common engineering technical personnel in this area make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determine.
Sequence table
<110> China National Academy of Food & Fermentation Industries
The method of L MALIC ACID produced by <120> bio-transformation fumaric acid
<160> 1
<170> PatentIn version 3.3
<210> 1
<211>482
<211>DNA
<213> Clavispora lusitaniae yeast CICC 33054(Clavispora lusitaniae CICC 33054)
<400>1
gctcaaattt gaaatcctgc gggaattgta atttgaaggt ttcgtggtct gagtcggccg 60
cgcccaagtc cattggaaca tggcgcctgg gagggtgaga gccccgtatg gcgcacgccg 120
actctttgca ccgcggctcc gacgagtcga gttgtttggg aatgcagctc taagtgggtg 180
gtaaattcca tctaaagcta aatattggcg agagaccgat agcgaacaag tacagtgatg 240
gaaagatgaa aagcactttg aaaagagagt gaaacagcac gtgaaattgt tgaaagggaa 300
gggcttgcaa gcagacacgg ttttaccggg ccagcgtcga aaagggggga ggaacaagaa 360
ctcgagaatg tggcgcgcac cttcgggcgc gcgtgttata gctcgtgttg acgcctccat 420
cccttttcga ggcctgcgat tctaggacgc tggcgtaatg gttgcaagcc gcccgtcttg 480
aa 482
Claims (8)
1. a strain Clavispora lusitaniae yeast CICC 33054(Clavispora lusitaniae CICC 33054), deposit number is CGMCC No.9848.
2. Clavispora lusitaniae yeast CICC 33054(Clavispora lusitaniae CICC 33054 as claimed in claim 1) produce L MALIC ACID for bio-transformation fumaric acid.
3. the method for L MALIC ACID produced by bio-transformation fumaric acid as claimed in claim 2, it is characterized in that Clavispora lusitaniae yeast CICC 33054 fermentation culture according to claim 1, centrifugal yeast cell, yeast cell adds sodium fumarate solution and carries out bio-transformation, through Fumaric acid, calcium salt cementation, filtration, sulfuric acid solution, filtration, concentrated, crystallization, dry L MALIC ACID finished product.
4. method according to claim 3, is characterized in that comprising the following steps:
(1) Clavispora lusitaniae yeast CICC 33054 slant strains inoculation seed culture medium fermentation culture 18 ~ 24h, obtains inoculation liquid;
(2) inoculation liquid access fermention medium, fermentation 20 ~ 30h obtains fermented liquid, fermented liquid centrifugal acquisition Clavispora lusitaniae yeast CICC 33054 cell;
(3) Clavispora lusitaniae yeast CICC 33054 cell adds sodium fumarate solution, and bio-transformation generates L MALIC ACID, through Fumaric acid, calcium salt cementation, filtration, sulfuric acid solution, filtration, concentrated, crystallization, dry L MALIC ACID finished product.
5. as claim 4 the method told, it is characterized in that step (1) and (2) Clavispora lusitaniae yeast CICC 33054 culture temperature are 25-35 DEG C.
6. as claim 4 the method told, it is characterized in that step (2) fermention medium composition (g/L) is: glucose 50.0 ~ 120.0, yeast powder 2.0 ~ 8.0, ammonium sulfate 3.0 ~ 8.0, potassium primary phosphate 1.0 ~ 5.0, magnesium sulfate heptahydrate 0.1 ~ 0.6, adjust ph 6.0 ~ 7.0.
7. as claim 4 the method told, it is characterized in that step (3) sodium fumarate solution ph is 6.0 ~ 8.0.
8. as claim 4 the method told, its special medical treatment is step (3) bio-transformation temperature 30 ~ 45 DEG C.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108300740A (en) * | 2017-01-13 | 2018-07-20 | 山东阜丰发酵有限公司 | A method of preparing L MALIC ACID |
| CN110438019A (en) * | 2019-06-06 | 2019-11-12 | 浙江工业大学 | A kind of composite bacteria agent and its fermented garbage from kitchen prepare the application of organic liquid fertilizer |
| CN111363686A (en) * | 2020-03-05 | 2020-07-03 | 湖南农业大学 | Yeast for brewing aromatized wine and application and method thereof |
| CN111732506A (en) * | 2019-07-02 | 2020-10-02 | 安徽丰原发酵技术工程研究有限公司 | Method for separating and extracting high-purity malic acid |
| CN111909025A (en) * | 2019-07-02 | 2020-11-10 | 安徽丰原发酵技术工程研究有限公司 | Method for producing calcium hydrogen malate and application of method in production of malic acid |
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| CN108300740A (en) * | 2017-01-13 | 2018-07-20 | 山东阜丰发酵有限公司 | A method of preparing L MALIC ACID |
| CN108300740B (en) * | 2017-01-13 | 2021-08-03 | 山东阜丰发酵有限公司 | Method for preparing L-malic acid |
| CN110438019A (en) * | 2019-06-06 | 2019-11-12 | 浙江工业大学 | A kind of composite bacteria agent and its fermented garbage from kitchen prepare the application of organic liquid fertilizer |
| CN111732506A (en) * | 2019-07-02 | 2020-10-02 | 安徽丰原发酵技术工程研究有限公司 | Method for separating and extracting high-purity malic acid |
| CN111909025A (en) * | 2019-07-02 | 2020-11-10 | 安徽丰原发酵技术工程研究有限公司 | Method for producing calcium hydrogen malate and application of method in production of malic acid |
| CN111732506B (en) * | 2019-07-02 | 2022-07-12 | 安徽丰原发酵技术工程研究有限公司 | Method for separating and extracting high-purity malic acid |
| CN111909025B (en) * | 2019-07-02 | 2022-07-15 | 安徽丰原发酵技术工程研究有限公司 | Method for producing calcium hydrogen malate and application of method in production of malic acid |
| CN111363686A (en) * | 2020-03-05 | 2020-07-03 | 湖南农业大学 | Yeast for brewing aromatized wine and application and method thereof |
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| CN104357340B (en) | 2017-08-01 |
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